Claims
- 1. A method for identifying a transformed cell which has undergone site-specific homologous recombination utilizing a AMFP vector comprising:
a) transforming cells with a AMFP vector designed to undergo site-specific homologous/recombination wherein the vector comprises:
a first DNA sequence which is substantially homologous to an endogenous genomic sequence present within the host genome; a second DNA sequence which encodes a fluorescent protein selection characteristic lacking regulatory elements and sequences coding for an initiating methionine in said cells and is non-homologous to cellular endogenous genomic sequences and therefore incapable of undergoing site-specific homologous recombination; a third DNA sequence which is substantially homologous to an endogenous genomic sequence present within the host genome and is different from the first DNA sequence; and b) propagating cells to select for or enrich for those which have been transformed with said AMFP vector by selecting for the presence of the functional fluorescent protein selectable marker gene product of said second DNA sequence; and c) separating cells which have said second DNA sequence encoding a functional fluorescent protein selectable marker from cells which do not have said second DNA sequence.
- 2. The method of claim 1, further comprising d) characterizing the genomic DNA of said cells carrying the second DNA sequence encoding a functional fluorescent protein selectable marker for the site-specific homologous recombination events which allow for modification of the cellular target DNA.
- 3. The method of claim 1 wherein said AMFP vector includes selectable markers which may be detected by fluorescence light emission.
- 4. The method of claim 1 wherein said fluorescent protein selectable markers allow for the separation of cells containing DNA encoding one marker from cells containing DNA encoding another or both markers.
- 5. The method of claim 1 wherein said cells are capable of homologous recombination.
- 6. The method of claim 1 wherein said cells are from a multicellular organism.
- 7. The method of claim 1 wherein said cells are from plants.
- 8. The method of claim 1 wherein said cells have undergone multiple rounds of site-specific homologous recombination for the purposes of multiple modifications of the endogenous cellular genome.
- 9. The method of claim 1 wherein said cells may be utilized to create a multicellular organism.
- 10. The method of claim 1 wherein said cells are embryonic stem cells.
- 11. An isolated AMFP gene targeting vector for site-specific homologous recombination in cells capable of undergoing homologous recombination, the vector comprising:
a first DNA sequence which is substantially homologous to cellular endogenous genomic sequences and is capable of undergoing homologous recombination in said cells, a second DNA sequence which is nonhomologous to cellular endogenous genomic sequences, is not capable of undergoing homologous recombination in said cells, does not contain regulatory elements, encodes a fluorescent protein selectable marker lacking sequences coding for an initiating methionine and capable of allowing for the identification of cells containing said positive selectable marker, a third DNA sequence which is substantially homologous to cellular endogenous genomic sequences and is capable of undergoing homologous recombination in said cells,
wherein the organization of said AMFP gene targeting vector in 5′ to 3′ orientation comprises: the first DNA sequence which is substantially homologous to cellular endogenous genomic DNA sequences, the second DNA sequence which does not contain regulatory elements and encodes a fluorescent protein selectable marker which lacks sequences coding for an initiating methionine, and the third DNA sequence which is substantially homologous to cellular endogenous genomic DNA sequences; wherein the vector is capable of undergoing site-specific homologous recombination resulting in modification of cellular endogenous target genomic DNA sequences.
- 12. The AMFP gene targeting vector of claim 11 wherein said cellular endogenous genomic target DNA is comprised of exons and introns.
- 13. The AMFP gene targeting vector of claim 11 wherein said vector contains all or portions of exons and introns which are substantially homologous to cellular target genomic DNA sequences.
- 14. The AMFP gene targeting vector of claim 11 wherein said vector contains portions of regulatory elements which are substantially homologous to cellular target genomic DNA sequences.
- 15. The AMFP gene targeting vector of claim 11 wherein said vector contains alterations in sequences which are substantially homologous to cellular target genomic DNA sequences including deletions, substitutions, additions or point mutations.
- 16. The AMFP gene targeting vector of claim 11 wherein said fluorescent protein selectable marker encoded by said second DNA sequence is selected from DNA sequences encoding fluorescent proteins including GFP, CFP, YFP, RFP, dsRED or HcRED.
- 17. The AMFP gene targeting vector of claim 11 wherein said vector has lengths of homology for said first and third DNA sequences which are between about 50 bp and 50,000 base pairs.
- 18. The AMFP gene targeting vector of claim 11 wherein said vector results in the modification of cellular endogenous genomic target DNA sequences.
- 19. The AMFP gene targeting vector of claim 11 wherein said vector introduces exogenous regulatory elements into the cellular endogenous genomic target DNA sequences.
- 20. An enriched population of cells generated through a method according to claim 1 wherein said cells have undergone site-specific homologous recombination.
- 21. A non-human transgenic animal generated by the method of claim 1 wherein said animal has been generated from cells which have undergone site-specific homologous recombination.
- 22. A transgenic plant generated by the method of claim 1 wherein said plant has been generated from cells which have undergone site-specific homologous recombination.
RELATED APPLICATION DATA
[0001] This application claims the benefit of priority to under 35 U.S.C. § 119(e)(1) of U.S. Ser. No. 60/348,549, filed Jan. 14, 2002, the entire context of which is incorporated herein by reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60348549 |
Jan 2002 |
US |